Striatal Neurons In Primary Culture Research Articles

Quisqualate agonists occlude kainate-induced current in cultured striatal neurons

We employed the whole cell patch-clamp technique to examine the ionic currents induced via activation of kainate/quisqualate receptors on striatal neurons in primary culture when N-methyl- d-aspartate receptors were blocked by selective antagonists. Bath perfusion of 10 μM-1 mM each of quisqualate, glutamate, α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (a selective quisqualate agonist) or kainate, induced only a sustained current, but more rapid application by pressure ejection of each of the first three agonists (but not kainate) also activated a rapidly desensitizing current. The current induced by a near-saturating concentration of kainate (1 mM) was, on average, 16-fold larger than the maximum sustained current induced by quisqualate (10 μM), or 7.5-fold larger than that induced by a-amino-3-hydroxy-5-methyl-4-isoxazole propionate (100 μM) or glutamate (100 μM). When kainate (100 μM-10 mM) was co-applied with each of the agonists (1 μM-1 mM), the sustained current was not the algebraic sum of the currents activated by kainate or the other agonist alone; rather, the kainate-induced current was increasingly occluded by co-application with increasing concentrations of another agonist. The potency to occlude kainate-induced current had a rank order of quisqualate > α-amino-3-hydroxy-5-methyl-4-isoxazole propionate ≈ glutamate; although at sufficiently high concentrations all three agonists could occlude the kainate-induced current completely. When kainate and quisqualate were co-applied during the continued presence of quisqualate, the onset of the kainate-induced sustained current was dramatically slowed. However, the steady-state occlusion by quisqualate could be abolished when the ratio kainate to quisqualate was raised to 100:1; therefore, the occlusion appears to involve a competition between kainate and quisqualate at some shared receptor binding sites which have a higher affinity for quisqualate than kainate.

Dopamine-induced homologous and heterologous desensitizations of adenylate cyclase-coupled receptors on striatal neurons

Striatal neurons in primary culture express a wide variety of adenylate cyclase-coupled receptors, and particularly dopamine (DA) D 1 receptors, which are known to induce an increase in cyclic AMP production. To study desensitization of those receptor-mediated responses, neurons were incubated in the presence of saturating concentrations of DA for various times. We observed a rapid desensitization of the D 1 response after 15 min, which reached a maximum after 18 h. This effect is reversible since incubation of treated neurons in fresh medium led to a complete recovery of the dopamine response within 2 days. In addition, a brief treatment with DA resulted in heterologous desensitization of β-adrenoceptors and 5-HT and vasoactive intestinal peptide (VIP) receptors coupled to adenylate cyclase on striatal neurons. Similar to what was observed for homologous desensitization, these effects are obtained within the first 15 min of exposure to DA; however, they are short-lasting, even in the persistent presence of DA.

Establishment of a long-term primary culture of striatal neurons

A new method of obtaining long-term primary cultures (lasting more than 8 weeks) of striatal neurons is described in this paper. The originality of the method consists of: (1) starting the culture for 3 days in a serum-free medium which allows attachment and neurite proliferation of neurons as well as the death of non-neuronal cells (mainly consisting of astrocytes); (2) introducing a limited amount of fetal calf serum (FCS) (2–5%) after 3 days in vitro (3 DIV), which likely provides optimal neuronal survival and attachment factors, and a limited amount of astrocyte proliferating factors. The period of introduction of serum, as well as the amount of serum introduced are critical factors. By phase contrast and transmission electron microscopy, we observed that neurons continued to develop neurite extensions, synaptic vesicles and synapse formations up to 50 DIV. Neuronal membranes, and synaptic contacts were particularly healthy up to 50 DIV. Interestingly, the number of astrocytes was constant between 30–50 DIV and limited to about 10%. We therefore obtained an equilibrium between neuronal and astrocyte differentiation and proliferation. It is likely that the small population of astrocytes, plus the low percentage of FCS added, provide essential factors for neuronal survival and differentiation, whereas a high density of differentiated neurons inhibited astrocyte cell proliferation. The clear-cut stability of these neuronal cultures goes in parallel with the stability of the pharmacological responses studied here: the coupling of carbachol and quisqualate receptors with the inositol phosphate production system. The culture method described here could be of particular interest to pursue biochemical, pharmacological and biological studies on neurons as well as on reciprocal interactions between neurons and astrocytes.

Kainate evokes the release of endogenous glycine from striatal neurons in primary culture

The actions of 56 mM KCl and excitatory amino acid (EAA) agonists on the release of endogenous glycine (Gly) from striatal neurons in primary culture was examined. During a 3 min period, 2 × 10 6 striatal neurons released 743 ± 51 pmol of Gly. In the presence of 56 mM KCl, an additional 492 ± 52 pmol of Gly (+ 66%) were released, 75% of which was dependent upon the presence of extracellular calcium. When striatal neurons were exposed to 1 mM N- methyl- d-aspartate (NMDA) or quisqualate (QA), endogenous Gly released was increased by 370 ± 71 (+ 50%) or 120 ± 31 (+ 16%) pmol, respectively. In the presence of 1 mM kainate (KA), however, the release of endogenous Gly increased by 994 ± 82 pmol (+ 135%). Interestingly, while KA (1 mM) was twice as effective as KCl (56 mM) in evoking the release of endogenous Gly, KCl was 5 times more effective than KA in evoking the release of endogenous γ-aminobutyric acid (GABA). KA-induced increases of endogenously released Gly were dose-dependent (EC 50, 100 μM), saturable and not significantly reduced in the absence of extracellular calcium. The actions of KA were blocked by coincubation with 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline (CNQX), a competitive antagonist at the KA receptor. These data suggest that the release of endogenous Gly from striatal neurons in primary culture is regulated principally by EAA actions at the KA receptor system.

Two distinct quisqualate receptor systems are present on striatal neurons

At concentrations at which it did not alter spontaneous release, quisqualate (QUIS) induced a dose-dependent (EC 50, 0.5 μM) potentiation of KCl- or veratrine-evoked release of [ 3H]GABA from striatal neurons in primary culture. QUIS potentiation of KCl-evoked [ 3H]GABA release was mimicked by the selective agonist α-amino-3-hydroxy-5-methylisoxazole-propionic acid (AMPA), glutamate and kainate, and was blocked by kynurenic acid and γ- d-glutamyglycine. QUIS also induced a dose-dependent (EC 50, 0.2 μM) augmentation of [ 3H]inositol monophosphate production in striatal neurons. This action of QUIS was mimicked by glutamate, but not by AMPA nor by kainate. Furthermore, none of the antagonists tested (kynurenic acid, γ- d-glutamylglycine, glutamic acid diethyl ester, and 4-aminophosphonobutanoic acid) could block QUIS-induced elevations in [ 3H]inositol monophosphate production. The results of the present study suggest that two QUIS receptor systems, distinguished on the basis of their pharmacological properties, may subserve specific roles in the regulation of striatal neuron function by excitatory amino acids.

Complex interaction between quisqualate and kainate receptors as revealed by measurement of GABA release from striatal neurons in primary culture

The effects of non-NMDA receptor agonists were tested on endogenous GABA and [ 3H]GABA release from highly purified striatal neurons differentiated in primary culture. Kainate (KA), glutamate (Glu) and quisqualate (QA) stimulated [ 3H]GABA release with EC 50s = 85 ± 20 (n = 6), 6.21 ± 1.42 (n = 3) and 0.135 ± 0.035 (n = 3) μM, respectively. KA was the most potent (in term of efficacy) agonist (maximal response at 10 mM: 935 ± 51% (n = 6) increase over basal release) followed by Glu (at 100 μM: 404 ± 34% (n = 5) increase) and QA (at 10 μM: 91 ± 6% (n = 6) increase). Phencyclidine (PCP), which was without effect on QA- and KA-evoked GABA release, inhibited the Glu response by about 50%. QA totally inhibited KA (50 μM)-evoked GABA release with an IC 50 = 0.39 ± 0.11 (n = 4) in a competitive manner (K i = 0.39 ± 0.07 μM (n = 3)). Competitive inhibition of the KA response was also observed with the other agonists of the quisqualate receptor, Glu and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), suggesting that Glu, QA and AMPA act as partial agonists at the KA receptor. γ-D-Glutamylaminomethylsulfonic acid (GAMS) also inhibited (IC 50 = 2.1 mM) the KA response competitively. However the inhibition by GAMS and QA was not additive. The response to QA was rapidly inactivated (no response after 3 min stimulation) in contrast to the KA-evoked GABA release which remained maximal for at least 3 min. When neurons were first exposed to concavanalin A (con A), a lectin known to inhibit Glu receptor desensitisation on insect muscles, the QA response remained maximal for at least 6 min. Con A greatly enhanced the maximal responses to QA and AMPA and decreased their apparent affinities. The KA-evoked GABA release (but not the veratridine and NMDA effects) was also augmented (no change in the EC 50 value) by con A. It is proposed that QA, AMPA and KA act at the same receptor-channel complex (termed G 2 receptor) which is desensitised more rapidly when stimulated by QA or AMPA than when stimulated by KA.

Translocation and Activation of Protein Kinase C in Striatal Neurons in Primary Culture: Relationship to Phorbol Dibutyrate Actions on the Inositol Phosphate Generating System and Neurotransmitter Release

The actions of the tumor-promoting phorbol ester phorbol dibutyrate were examined, under identical physiological conditions, on three distinct cellular processes in striatal neurons: the distribution of protein kinase C, the carbachol-stimulated generation of [3H]inositol monophosphate, and the KCl-evoked release of gamma-[3H]aminobutyric acid ([3H]GABA). Phorbol dibutyrate induced a rapid (complete in 5 min), dose-dependent, entirely reversible (t0.5 = 15 min) translocation of protein kinase C from cytosol to membrane. On longer exposure to phorbol dibutyrate, membrane-associated protein kinase C returned toward the control level, and total cellular enzyme activity declined markedly. Phorbol dibutyrate also induced the dose-dependent attenuation of carbachol-stimulated [3H]inositol monophosphate production and potentiation of KCl-evoked release of [3H]GABA. The translocation of protein kinase C and the potentiation of KCl-evoked [3H]GABA release were both rapidly reversed following washout of phorbol dibutyrate. In addition, for both processes, the effect of a 1-h exposure to phorbol dibutyrate was markedly less than that observed following a 5-min exposure to the agent. In direct contrast, inhibition of carbachol-stimulated [3H]inositol monophosphate production was not rapidly reversed following washout of phorbol dibutyrate and was actually more pronounced following a 1-h exposure, compared with a 5-min exposure. These findings indicate that some, but not all, of the actions of phorbol dibutyrate are closely associated with the translocation of protein kinase C in striatal neurons in primary culture.

Two distinct mechanisms, differentially affected by excitatory amino acids, trigger GABA release from fetal mouse striatal neurons in primary culture

The mechanisms leading to Ca2+-dependent and Ca2+-independent GABA release were studied on highly purified striatal neurons developed in primary culture. Ca2+-dependent GABA release, which represents about 75% of the 56 mM K+ effect was totally inhibited when striatal neurons were first exposed to tetanus toxin (TnTx) (10 micrograms/ml) for 24 hr. The K+ effect was potentiated when 1 mM nipecotic acid (an inhibitor of the GABA uptake system) was added during the stimulation period or when Na+ was replaced by Li+. However, no difference in the GABA release measured under high-K+ conditions was observed after a 22 min preincubation of the neurons in a medium containing nipecotic acid or Li+. Replacement of Cl- ions by SO4(2-) did not modify K+-evoked GABA release. Ca2+-independent GABA release was stimulated by veratridine (20 microM), ouabain (3 mM), and monensin (20 microM), as well as the excitatory amino acids glutamate (100 microM), N-methyl-D-aspartate (100 microM), quisqualate (10 microM), and kainate (1 mM), drugs known to increase intracellular Na+ concentration. The veratridine- or glutamate-evoked GABA release was neither inhibited when intracellular Ca2+ content was reduced by more than 90% nor by treatment of the neurons to TnTx. However, the Ca2+-independent GABA release elicited by veratridine was inhibited by preincubation of the neurons in a medium containing 1 mM nipectotic acid and in a medium containing Li+ instead of Na+ or SO4(2-) instead of Cl-. These results strongly suggest that 2 different GABA release mechanisms exist in striatal neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Forskolin attenuates the evoked release of [ 3H]GABA from striatal neurons in primary culture

The actions of the diterpene forskolin, and cyclic AMP analogues, on the evoked release of [ 3H]GABA (γ-aminobutyric acid) was examined in intact striatal neurons in primary culture, generated from the fetal mouse brain. Exposure of striatal neurons to forskolin (100 μM) resulted in a 40–55% attenuation of [ 3H]GABA release evoked by either KCl (30 mM) or veratrine (2 μg/ml), while baseline levels of release were unaffected. The dose-dependence for forskolin in striatal neurons. Exposure of striatal neurons to membrane-permeable identical to the dose-dependent elevation of cyclic AMP levels by forskolin in striatal neurons. Exposure of striatal neurons to membrane-permeable analogues of cyclic AMP, such as p-chlorophenylthio cyclic AMP (0.5 mM) and dibutyryl cyclic AMP (1 mM), resulted in a 25 and 26% attenuation of [ 3H]GABA release, respectively; dibutyryl cyclic GMP (1 mM) was without effect. The similarity between the actions of forskolin and the cyclic AMP analogues suggests that, in striatal neurons in primary culture, the elevation of cyclic AMP levels results in the attenuation of the evoked release of [ 3H]GABA. The greater effectiveness of forskolin, compared to the cyclic AMP analogues, may be related to the recently reported, additional direct actions of forskolin on neuronal membrane ion channels.

Excitatory amino acid-evoked release of gamma-[3H]aminobutyric acid from striatal neurons in primary culture.

The actions of excitatory amino acids on the release of previously incorporated gamma-[3H]aminobutyric acid ([3H]GABA) were examined in purified (greater than 93%) striatal neurons derived from the fetal mouse brain and differentiated in primary culture. Glutamate, KCl, and veratrine evoked a dose-dependent, saturable, and reversible release of [3H]GABA from striatal neurons. Glutamate actions were not reduced in the absence of calcium, and were insensitive to tetrodotoxin. The dose-response relationships of excitatory amino acids demonstrated the following rank order of potency: glutamate greater than aspartate = N-methyl-D-aspartate greater than kainate much greater than quisqualate. Kainate, however, was the most effective agonist, evoking an eightfold increase over baseline levels of [3H]GABA release. Aspartate- and N-methyl-D-aspartate-evoked release was abolished in the presence of either 2-aminophosphonovaleric acid or gamma-D-glutamylglycine. Release due to glutamate and kainate was partially or ineffectively attenuated by these agents. Glutamate-, aspartate-, and N-methyl-D-aspartate-evoked GABA releases were augmented when calcium was omitted from the bathing medium and reduced when sodium was replaced with choline or lithium. Kainate-evoked release was unaffected when calcium was omitted, virtually unchanged when choline replaced sodium, and markedly potentiated when lithium was substituted for sodium. These findings suggest that at least two distinct receptor systems for excitatory amino acids mediate the evoked release of [3H]GABA from striatal neurons in primary culture. These two systems, aspartate/N-methyl-D-aspartate- and kainate-preferring, are distinguishable on the basis of their pharmacological and ionic properties.

α 2-Adrenergic receptors mediate inhibition of cyclic AMP production in neurons in primary culture

The actions of adrenergic agents on the intracellular production of cyclic adenosine monophosphate (AMP) was examined in intact cortical and striatal neurons in primary culture, generated from the fetal mouse brain. Exposure of striatal neurons to the β-adrenergic agonist isoproterenol (10 μM) resulted in a 5-fold increase in intraneuronal cyclic AMP; norepinephrine (100 μM), alone or in combination with isoproterenol, produced only a 3-fold increase in cyclic AMP levels. However, in the presence of yohimbine (10 μM), cyclic AMP productions due to norepinephrine or isoproterenol plus norepinephrine were identical to isoproterenol alone. When striatal or cortical neurons were exposed to pertussis toxin (100 ng/ml) overnight, there was no detectable difference between isoproterenol- and norepinephrine-stimulated cyclic AMP production. These data suggest that α 2-adrenergic receptors mediate the attenuation of cyclic AMP production in neurons and do so via the inhibitory guanine nucleotide regulatory protein of adenylate cyclase.

Release of Endogenous Amino Acids from Striatal Neurons in Primary Culture

Endogenous amino acid release was examined in highly purified striatal neurons obtained from fetal mouse brain, and differentiated in primary culture. This study aimed to determine which amino acids are released from striatal neurons after a brief depolarization period induced by elevated potassium concentration or veratrine. Amino acids released into the extracellular medium, subsequent to a 3-min exposure of striatal neurons, were subjected to HPLC analysis. At 14 days in vitro potassium (56 mM) depolarization elicited a 25-fold increase in gamma-aminobutyric acid release, 85% of which was calcium-dependent. This effect was small but apparent at 7 days in vitro (two-fold increase) and greatly increased between 11 and 14 days in vitro, subsequent to the appearance of synaptic vesicles in nerve terminals. gamma-Aminobutyric acid release was readily reversible within minutes of return to the resting state. Veratrine induced a quantitatively similar but calcium-independent increase in gamma-aminobutyric acid release. Similar results were observed on aspartate and glutamate release, but the increase was very small even after 14 days in vitro (62.2 and 123.3% increase over basal release, respectively). Taurine and hypotaurine release increased during and after depolarization induced by potassium. This effect remained constant between 11 and 18 days in vitro. BAY K 8644, a dihydropyridine-sensitive calcium channel agonist, augmented the effect of 15 mM potassium on gamma-aminobutyric acid release, but this effect remained very small as compared to the potassium (56 mM) or veratrine effects. In addition, nifedipine inhibited this BAY K 8644-induced release. These results demonstrate the high level of differentiation among striatal neurons containing gamma-aminobutyric acid in this in vitro system.

Vasoactive intestinal peptide actions on cyclic AMP levels in cultured striatal neurons

The actions of vasoactive intestinal peptide (VIP) on intracellular cyclic AMP, in primary cultures of striatal neurons, were examined. VIP stimulated cyclic AMP formation five-fold over basal levels in neurons after 6 days in vitro (DIV); half maximal activation (EC 50) was obtained with 10 nM of the peptide. VIP stimulation was both more potent and effective than those due to adrenocorticotropin (ACTH), dopamine (DA) or serotonin (5-HT). VIP efficacy was augmented to 15–20-fold in the presence of 0.1 μM forskolin, which had virtually no effect on cyclic AMP production alone; VIP potency was unaffected. At saturating concentrations of VIP (0.1–1.0 μM), no other agonist can further activate cyclic AMP production. Under these conditions, the interaction with opiate, DA D 2 and 5-HT 1 receptors, whose activation results in the inhibition of cyclic AMP production, was shown. During the differentiation of striatal neurons, VIP stimulation of cyclic AMP over basal levels, in the presence of 0.1 μM forskolin, decreases progressively from 30-fold after 3 DIV to 11-fold after 10–13 DIV.